Abberant kinase-depenent signaling is associated with the etiology of several cancers. For this reason, pharmacological agents are being developed to modulate kinase-dependent signaling as potential new anticancer therapeutics. We are developing kinase-dependent signaling inhibitors that function by: (1) Blocking protein-protein associations mediated by recognition and binding of the polobox binding domain (PBD) of polo-like kinase 1 (Plk1) to phosphothreonine (pThr)/phosphoserine (pSer)-containing protein sequences and (2) Blocking the removal of phosphoryl groups by cellular protein-tyrosine phosphatases (PTPs). Overexpression of the serine/threonine polo-like kinase 1 (Plk1) is tightly associated with oncogenesis in several human cancers. Interference with Plk1 function induces apoptosis in tumor cells but not in normal cells. Accordingly, Plk1 is a potentially attractive anticancer chemotherapeutic target. Plk1 possesses a unique phosphopeptide binding polo box domain (PBD) that is essential for its intracellular localization and mitotic functions. Unlike kinase domains, PBDs are found only in the four members of Plks. Therefore, they represent ideal targets for selectively inhibiting the function of Plks. By examining various PBD-binding phosphopeptides, our NCI collaborator, Dr. Kyung Lee, previously found that the 5-mer phosphopeptide PLHSpT specifically interacts with the Plk1 PBD with high affinity, whereas it fails to significantly interact with the PBDs of two closely-related kinases, Plk2 and Plk3. Starting from this peptide, we employed an iterative sequential process of structural refinement to arrive at new agents, which bind with high affinity to the Plk1 PBD. Several of these agents can inhibit binding interactions with the Plk1 PBD at concenterations (low nanomolar) that are 10,000-times more potent than the parent PLHSpT peptide. These peptides retain high selectivity for the Plk1 PBD relative to the related Plk2 or Plk3 PBDs. In collaboration with Dr. Michael Yaffe (MIT) X-ray co-crystal structures of these peptides bound to Plk1 PBD indicate unanticipated modes of binding that take advantage of a cryptic binding channel that is not present in the non-liganded PBD. Although critical elements in the high affinity recognition of peptides and proteins by PBD are derived from pThr/pSer-residues, the use of these residues in therapeutics is potentially limited by poor cellular uptake, in part due to high anionic charge of the phosphoryl moiety. We have recently discovered new synthetic transformations that lessen the overall peptide anionic charge by intramolecular charge masking, which provides peptides with enhanced efficacy in cellular assays. In further work we are developing proteins that merge properties of antibodies with biologically active small molecules. This work is being done in collaboration with Dr. Christoph Rader (Scripps Florida). Our approach employs monoclonal antibodies and antibody Fc fragments harboring a single C-terminal selenocysteine residue (Fc-Sec). The resulting antibody drug conjugates (ADCs) are directed against a variety of targets by changing the peptide or small molecule to which they are conjugated. In one aspect of our work, we have employed a variety of chemistries to attach biologically-cleavable linkers that allow release of cargo once delivery to the target has been achieved. We have developed versatile hetero-bifunctional linkers incorporating biologically cleavable bonds that are compatible with multiple types of Cu-free Huisgen 1,3-dipolar cycloaddition reagents. These linkers contain both targeting functionality and drug payloads. In one aspect of our work involving the potently cytotoxic peptide, monomethyl auristatin F (MMAF), we are examining linkers that can be conjugated to the Fc-Sec protein by nucleophilic alkylation reactions. This work has involved developing new synthetic routes to key components of the MMAF peptide.